Association between ischemia-modified albumin (IMA) and peripheral endothelial dysfunction in rheumatoid arthritis patients

The identification of circulating biomarkers of endothelial dysfunction (ED), a precursor to atherosclerosis, in rheumatoid arthritis (RA) would facilitate early risk stratification and prevention strategies. Ischemia-modified albumin (IMA) has emerged as a potential biomarker of oxidative stress, ischemia, and ED. However, studies examining the relationship between IMA and ED in RA patients are lacking. We measured serum IMA concentrations by using an albumin cobalt binding test and peripheral vasodilatory capacity by EndoPAT in 113 RA patients without previous cardiovascular events enrolled in the EDRA study (ClinicalTrials.gov: NCT02341066). The mean peripheral vasodilatory capacity, expressed by the log of reactive hyperemia index (logRHI), was 0.82, corresponding to 27% RA patients having ED. The mean plasma concentrations of IMA were 0.478 absorbance units. We observed a significant and inverse association between peripheral vasodilatory capacity and serum IMA concentrations (rho =  − 0.22, p = 0.02). In univariate logistic regression, ED was significantly associated with serum IMA concentrations [OR 1173 (95% CI 1.3568 to 101,364), p = 0.040) and higher disease activity. In multivariate logistic regression, the independent association between ED and IMA remained significant after correction for disease activity and other RA-confounders [OR 2252 (95% CI 1.0596 to 4,787,505), p = 0.048 in Model 1; OR 7221 (95% CI 4.1539 to 12,552,859), p = 0.02 in Model 2]. Conclusions: This study suggests that IMA is a promising biomarker of ED in RA. Further research is needed to confirm our findings and determine the clinical utility of IMA in detecting and managing early atherosclerosis in RA patients.

Ischemia modified albumin (IMA), resulting from chemical changes of albumin during ischemia, has been suggested as a candidate biomarker for acute coronary syndrome 16 .Several studies have also reported increased serum IMA concentrations in non-cardiac diseases (e.g., sepsis, intestinal ischemia, acute pulmonary embolism) as a result of a common background of acute and chronic inflammatory state, increased oxidative stress, and reactive oxygen species formation 17 .Moreover, IMA formation has been related to ED, the early step of atherosclerotic vascular disease 18 .
Elevated serum IMA concentrations have also been reported in RA patients compared to the general population and have been linked to inflammatory state, oxidative stress, and increased carotid intima-media thickness [19][20][21] .However, the possible link between IMA and ED in this patient group has not been investigated.
Therefore, we sought to address this issue by studying the relationship between serum IMA concentrations and disease activity and peripheral endothelial vasodilatory capacity in RA patients free of previous cardiovascular events.

Patients
Consecutive unselected RA patients fulfilling the 2010 European League Against Rheumatism and American College of Rheumatology classification criteria 22 were prospectively enrolled in the Bio-RA study between October 2015 and July 2017.The Bio-RA study is an ancillary study of the Endothelial Dysfunction Evaluation for Coronary Heart Disease Risk Estimation in Rheumatoid Arthritis study (ClinicalTrials.gov:NCT02341066).Inclusion and exclusion criteria for both studies have been previously reported 9,12 .
All participants in the Bio-RA and EDRA studies underwent a comprehensive assessment of traditional cardiovascular risk factors, including hypertension (defined as a blood pressure ≥ 140/90 mmHg or treatment with antihypertensive medications), diabetes (diagnosed according to the patient's history and/or treatment with insulin or oral hypoglycemic agents), dyslipidemia (defined according to either a recent lipid profile, the patient's history and/or treatment with hypolipidemic drugs), and smoking status.Additionally, data on current treatment with disease-modifying antirheumatic drugs (DMARDs) as well as laboratory parameters of inflammation, such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and neutrophil-to-lymphocyte ratio (NLR) 23,24 , Disease Activity Score-28 (DAS-28), positivity for Rheumatoid Factor (RF), and anticitrullinated cyclic peptide antibodies (ACPA), were collected.RA patients with DAS-28 ≤ 2.6 were considered in remission, while those with DAS-28˃2.6 were considered as having active disease.

Pulse amplitude tonometry
Peripheral vasodilatory capacity was assessed using a non-invasive and validated technique known as pulse amplitude tonometry (PAT) measured using the EndoPAT 2000 system (Itamar Medical Inc., Caesarea, Israel).EndoPAT is an FDA-approved device that measures changes in peripheral arterial tone and pulsatile blood flow in response to reactive hyperemia.This technique is based on the principle that increased blood flow through an occluded artery leads to an increase in shear stress, which induces endothelium-dependent vasodilation.EndoPAT measures this response by assessing the ratio of pulse amplitude after reactive hyperemia to the baseline pulse amplitude.A reactive hyperemia index (RHI) is calculated as the ratio of the post-occlusion pulse amplitude to the baseline pulse amplitude, and the logarithmic transformation of this index (lnRHI) is used as a marker of peripheral vasodilatory capacity with values of lnRHI ≤ 0.51 used as a marker of ED 25 .

Ischemia-modified albumin
Blood samples were collected using blood evacuation tubes containing EDTA (Vacutainer Systems Europe; Becton Dickinson, Meylan Cedex, France), on the same day of endothelial function evaluation and before EndoPAT testing.
Ischemia-modified albumin (IMA) was measured using an albumin cobalt binding test.50 μL of 0.1% cobalt (II) chloride (CoCl2, 6H2O) was added to the patient's serum and incubated for 10 min.Then, 50 μL of 1.5 mg/ mL dithiothreitol was added and incubated for 2 min after the mixing procedure.Finally, 1 mL of 0.9% NaCl solution was added to stop the reaction.A blank solution was prepared with the same method except for using distilled water instead of dithiothreitol.The absorbance of the samples was measured spectrophotometrically at 470 nm and results were expressed as absorbance units (ABSU).

Statistical analysis
Data are expressed as mean values (mean ± SD) or median values (median and IQR).The Kolmogorov-Smirnov test was performed to evaluate the variable distribution.Between-group differences of continuous variables were compared using unpaired Student's t-test or Mann-Whitney rank sum test, as appropriate.Kruskal-Wallis t-test has been used to evaluated difference in the IMA concentration across categories of increasing RA disease activity.Correlation analyses were performed by Pearson correlation test or Spearman's correlation rank test, as appropriate.
Predictors of ED were evaluated by univariate and multivariate logistic regression analysis.Multivariate analysis included confounders with p-values of < 0.2 in univariate analysis (DAS-28, ESR, NLR, ACPA and IMA).In case of collinearity between confounders, separate models were built by considering separate variables in each model.This is a pilot study, and a formal sample size analysis has not been performed.

Institutional review board statement
The Bio-RA and the EDRA studies were approved by the Ethics Committee of Azienda ASL 1 of Sassari (Italy) (2126/CE-2015 and 2219/CE-2015) and conducted in accordance with the Declaration of Helsinki.

Informed consent statement
Informed consent was obtained from all subjects involved in the study.
Despite not stated as a formal procedure in the protocol, when possible, a sex-balanced enrollment was performed with the aim of minimizing the effect of sex on measure of endothelial function and serum IMA concentrations.

IMA concentrations and RA disease activity
Mean serum IMA concentrations in the overall group of RA patients without previous cardiovascular events were 0.748 (0.718-0.778)ABSU.
Univariate logistic regression analysis (Table 2) confirmed a significant relationship between ED and serum IMA concentrations (OR 1173, 95% CI 1.36-1,013,645) and also demonstrated a significant association between ED and DAS-28 (OR 1.4899, 95% CI 1.0031 to 2.2129, p = 0.048).In multivariate logistic regression (Table 3), serum IMA concentrations remained significantly associated with ED after correction for confounders.

Discussion
In this study, we reported for the first time, the presence of a significant relationship between serum IMA levels and impaired peripheral vasodilatory capacity in patients with RA, that appears to be independent from common atherosclerotic cardiovascular risk factors.A range of distinctive potential mechanisms at play in RA patients, including ED, atherosclerosis and microthrombosis, may be supposed to underly this association.
ED has been demonstrated early in the natural history of RA 26 and seems to be related to chronic inflammatory burden, and increased prevalence of conventional cardiovascular risk factors with changes in lipid and glucose metabolism, metabolic syndrome, and side effect of specific immunosuppressive drugs having a crucial role 4,6,27 .Reduced peripheral vasodilatory capacity, may also depends on unbalanced autonomic nervous system control 9 in RA patients with immuno-autonomic dysfunctions 28,29 .Autonomic changes of microvascular tone, may favor repetitive cycles of ischemia-reperfusion, leading in turn to tissue damage.Therefore, in the setting of reduced vasodilatory response, raised IMA levels may represent the result of chronic endothelial damage, ischemia, and increase oxidative stress.In accordance with this hypothesis, IMA levels have been reported to be correlated to ED, measured by flow-mediated dilatation (FMD), in male diabetic patients with erectile dysfunction 30 .www.nature.com/scientificreports/Atherosclerotic process in autoimmune diseases and RA, has been reported to develop early in arterial walls and may recognize multiple mechanisms including inflammatory response, local breach of tolerance, and macrophages migration and accumulation underneath the endothelial layer.Atherosclerotic plaque may decrease local blood flow inducing a condition of chronic tissue hypoxia and increased oxidative stress.Occlusion of coronary vessels is acutely followed by an increase of serum IMA concentration in patients with coronary syndrome, but it is likely that also chronic hypoxia in atherosclerotic arterial beds may be associated to raised serum IMA concentrations.Accordingly, Uslu et al. 19 reported a significant correlation between serum IMA concentrations and carotid intima-media thickness in RA population.
Microthrombosis, linked to proinflammatory cytokines, pro-thrombotic autoantibodies 31 , and damaged endothelial barrier, may induce local tissue ischemia, oxidative stress and in increased albumin oxidation in RA.Accordingly, raised IMA levels have been reported in COVID-19, deep venous thrombosis, and pulmonary embolism 32,33 .www.nature.com/scientificreports/ In our study there was only a trend towards a significant association between serum IMA levels and RA disease activity.Despite chronic inflammation would be crucial in the development of ED, microthrombosis and atherosclerosis, it is difficult to find an association between current indirect measures of inflammation and serum IMA levels.Most of our patients were under immunosuppressive treatment and therefore we cannot rule out the confounding effect of specific drugs on the relationship between IMA and peripheral vasodilatory capacity [34][35][36][37] .
Our study has some limitations.First, we did not prospectively investigate whether serum IMA concentrations predict ischemic events in our RA population.Second, measures of endothelial function with FMD (a gold-standard biomarker of ED) and other invasive techniques would have strengthen EndoPAT findings and should be implemented in future research on this subject.Third, the lack of a control group does not allow to establish whether the observed correlation between IMA and ED is specific to RA.

Conclusions
Serum IMA concentrations are independently associated with impaired vasodilatory capacity and the presence of ED in RA patients free of overt cardiovascular disease.Further studies are needed to explore the mechanisms underlying this association and the potential role of IMA as a candidate biomarker for early atherosclerotic cardiovascular disease in RA.

Figure 1 .
Figure 1.Correlations between serum IMA concentrations and RA disease activity.

Table 2 .
Univariate logistic regression analysis assessing the association between patient characteristics and endothelial dysfunction.ACPA anticitrullinated cyclic peptide antibodies, BMI body mass index, CRP C-reactive protein, DAS-28 disease activity score-28, DMARDs disease-modifying antirheumatic drugs, ESR erythrocyte sedimentation rate, IMA ischemic modified albumin, Ln-RHI Log-transformed reactive hyperemia index, NLR neutrophils to lymphocytes ratio.In bold significant values.

Table 3 .
Multivariate logistic regression analysis assessing the association between patient characteristics and peripheral endothelial dysfunction.BMI body mass index, CRP C-reactive protein, Ln-RHI Logtransformed reactive hyperemia index, NLR neutrophils to lymphocytes ratio.ACPA Anticitrullinated cyclic peptide antibodies, DAS-28 disease activity score-28, DMARDs disease-modifying antirheumatic drugs, ESR erythrocyte sedimentation rate, IMA ischemic modified albumin, NLR neutrophils to lymphocytes ratio.Two models were created given the collinearity of DAS-28 and ESR.The models included all confounders showing associations with p < 0.2 in univariate logistic regression (ACPA, ESR, DAS28, IMA, NLR).Significant values are in bold.